Generating HPV antigen-specific cells from a naive T cell population

ABSTRACT

Safe, rapid and efficient methods for producing antigen-specific T cells recognizing human papilloma virus or HPV antigens.

CROSS-REFERENCE(S) TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 15/771,648,filed Apr. 27, 2018, which is the National Stage of InternationalApplication no. PCT/JP2016/059683, filed Oct. 31, 2016, which claimspriority to U.S. Provisional Application No. 62/248,818, filed Oct. 30,2015, which is incorporated by reference. This application is related toPCT/US2016/23413, filed Mar. 21, 2016 entitled “Generating virus orother antigen-specific cells for a naïve T cell population”, whichclaims priority to U.S. Provisional Application Nos. 62/135,851 and62/135,888, filed Mar. 20, 2015; and is related to PCT/US2014/62698,filed Oct. 28, 2014, entitled “Expansion of CMV-Specific T cells fromCMV-Seronegative Donors”, which claims priority to U.S. ProvisionalApplication No. 61/896,296, filed Oct. 28, 2013. All of the abovedocuments are incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This research was sponsored in part by a grant from the National CancerInstitute of the National Institutes of Health, grant #: NCI PO1CA148600-02 awarded to Catherine M. Bollard and Elizabeth J. Shpall.

BACKGROUND OF THE INVENTION Field of the Invention

The invention involves human papilloma virus- or HPV-antigen-specific Tcells and methods for producing them from the T cells of healthy ornaïve donors as well as a cell-based therapy using these HPV-specific Tcells for the treatment of diseases, disorders or conditions caused byor associated with human papilloma virus.

Description of the Related Art

Some current T-cell based immunotherapies use virus- and tumor-specificT cells expanded from samples containing T cells and precursor T cells.Virus-specific T cells have been shown to be effective against viralinfections after stem cell transplantation. T cell based cell therapiesusing virus-specific T-cell populations can provide protection fromvirus-infected cells and are associated with fewer side effects thanantiviral drugs.¹. T cell based therapies using expanded virus-specificpopulations have activity against virus-expressing malignancies^(2, 3)and induced a graft-versus-leukemia effect that cleared circulatingleukemic blasts.⁴

These immunotherapies have the advantage of providing lifelongprotection due to the generation of memory cell populations.³ Cells fromsamples having memory T cells are easily expanded ex vivo because thedonors from which they are derived have previously encountered thesepathogens, and hence, there are existing memory, virus-specific T cellsthat rapidly expand ex vivo.⁵ More importantly, studies have shown thata single HLA match is enough to mediate therapeutic efficacy of these Tcells, with minimal side effects. Therefore, banks of third party cellscan be generated and maintained, for off the shelf use of such products.

Unfortunately for certain viral infections and virus-associated tumorsnot all potential sources of these cells have been previously exposed tothe pathogen. One important example is HPV, which causes HPV infectionin immune compromised hosts⁶⁻⁸ and HPV-associated malignancies likecervical cancer.⁹⁻¹⁴ In the United States, seroprevalence for any of the9 HPV types is limited to approximately 40% for females and 20% formales. ¹⁵Most donors are therefore naïve to HPV antigens. This imposesobstacles to expansion of HPV-recognizing T cells from these samples dueto the absence of memory T cells.

It is difficult to expand antigen-specific T cells from these naïve cellpopulations, such as naïve T cells from unexposed/seronegative healthydonors or T cells from cord blood, because these T cells have never beenprimed by the antigen in vivo. Such populations lack antigen-specificmemory T cells that can be readily expanded. In other settings, proposedmethods for expanding such antigen-specific T cells from naïve donorscurrently use viruses, virus-infected cells, or virus-transformedcells.^(16, 17)

Methods that involve the use of viruses to produce antigen-specific Tcells for therapeutic use are undesirable because they are associatedwith increased clinical risks and significant regulatory hurdles. It isfor this reason that antigens in the form of peptides have been used toexpand antigen-specific T cells.¹⁸

Application of these peptide-based methods towards generatingHPV-specific T cells from naïve donors have met very limited success.¹²

Most successful attempts at generating T cells specific for HPV usedautologous cells from a patient who had been previously exposed to HPVantigens. In one such study, although more than 1,200-fold expansion wasseen, this was mostly limited to 8/156 cervical cancer patients and33/52 oropharyngeal cancer patients and only 1/20 healthy donors wherethe responding donor was believed to behave been previously exposed toHPV antigens.¹²

For third party bank applications, it is crucial to generateHPV-specific T cells from healthy donors regardless of previous exposureto HPV antigens.

BRIEF SUMMARY OF THE INVENTION

The method according to the invention advantageously permits the rapidand robust expansion of HPV-recognizing or HPV-specific T cells,provides T cells which recognize therapeutically important antigenstargeting opportunistic HPV infections and HPV-associated malignancies,and does not require the use of live viruses or virus-transformed cells.Here the inventors describe a new way of manufacturing these cells in aclinically-appliable way for use as a donor-derived and off-the-shelfproduct. The inventors leveraged their experience in generating T cellresponses from naïve cell sources ex vivo to manufacture HPV-recognizingor HPV-specific T cells. Based on the immunological properties of HPV,the inventors modified the process of generating antigen-presentingdendritic cells, modified the concentration of cytokines used forexpanding T cells, and used artificial APCs to help expand T cellnumbers.

In one of its embodiments, the invention provides a robust method forgenerating HPV antigen-specific T cells (or HPV antigen-recognizing Tcells). The generated HPV recognizing T cells may recognize a singleepitope of an HPV antigen, multiple epitopes of a single HPV antigen ormay recognize epitopes on different HPV antigens. The method employsoverlapping peptide libraries of HPV antigen(s) pulsed onto differentantigen presenting cells, such as dendritic cells, monocytes, K562cells, artificial antigen presenting cells, PHA blasts, B-blasts,lymphoblastoid cells, and CD3-28 blasts, different priming and expansioncytokines—including but not limited to IL2, IL7, IL15)—and differentselection methods (CD45RO depletion, etc)^(16, 17) Other artificial oralternative antigen presenting cells may also be used. Theantigen-specific T cells produced by the invention can be used to treatpost-transplant viral infections and tumor relapse. Theseantigen-specific T cells and their precursors can be advantageouslybanked or stored for later administration to a subject in need of T-cellimmunity against a particular pathogen or neoplastic condition.

The present invention comprises a process to specifically generate HPVantigen-specific T cells from the immune system of healthy donors, mostof whom will be HPV seronegative and thus have virus-naïve T cellpopulations. The invention is a process and its use aimed at preventingviral infections after transplant and in other immunocompromisedsettings and circumstances where HPV can be problematic, likeHPV-associated malignancies. The invention makesmulti-HPV-antigen-specific T cells from naïve T cells in aclinically-relevant way that has never been done before from naïve Tcells.

Because the invention itself is a process and use, it can be readilyapplied to generating antigen-specific responses from naïve populationsagainst other opportunistic viruses, such as, but not limited to, HHV6and BK virus. It can be expanded to include virus-specific antigens fromdiseases associated with malignancies such as but not limited to EBV andHIV. It can be combined with other cell products, lymphodepletingregimens, and epigenetic-modifying drugs. Other medical uses includepromoting engraftment and providing a therapy to immunodeficientpatients before a transplant.

The invention generates HPV-specific T cells using different overlappingpeptide libraries pulsed onto potentially different antigen presentingcells (dendritic cells, monocytes, K562 cells, PHA blasts, B-blasts,lymphoblastoid cells, and CD3-28 blasts), different priming andexpansion cytokines (including but not limited to IL2, IL7, IL15), anddifferent selection methods (CD45RO depletion, etc). Other artificial oralternative antigen presenting cells may be used as well. The inventorsalso describe a third party bank of the said cells manufactured fromnaïve T cells, along with the process of selecting the best match for adonor.

The process of the present invention is safe, simple, rapid andreproducible and can be used to produce HPV-recognizing or HPV-specificT cells in accordance with good manufacturing practices (GMP) for avariety of different patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . T cells that recognize HPV were generated using the methoddisclosed herein from healthy donors (n=5). They specifically recognizeHPV antigens E6 and E7 as compared to an irrelevant antigen (theself-protein actin). Background responses from cells alone (CTL) areshown. This shows that the process of the invention generates HPVspecific T cells from healthy donors.

FIG. 2 . T cells that recognize HPV (n=5) can be expanded to clinicallyrelevant numbers following three stimulations. This shows that theinvention is capable of robustly manufacturing HPV-recognizing orHPV-specific T cells for clinical or other uses.

FIG. 3 . T cells that recognize HPV (n=5) have a mixed phenotype (CD4and CD8), which has been shown to promote better responses from T cellinfusions. Cells also express the costimulatory receptors CD27 and CD28.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

“Accessory cell” is a cell, such as a K562 cell, that providescostimulation for recognition of peptide antigens by T cells or thatotherwise assists a T-cell recognize, become primed or expand in thepresence of a peptide antigen. Accessory cells may also be otherartificial or alternative antigen presenting cells.

An “activated T-cell” or “ATC” is obtained by exposing mononuclear cellsin cord blood or another sample containing naïve immune cells to amitogen, such as phytohemagglutinin (PHA) and/or Interleukin (IL)-2.

An “antigen” includes molecules, such as polypeptides, peptides, orglyco- or lipo-peptides that are recognized by the immune system, suchas by the cellular or humoral arms of the immune system. The term“antigen” includes antigenic determinants, such as peptides with lengthsof 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 ormore amino acid residues that bind to MHC molecules, form parts of MHCClass I or II complexes, or that are recognized when complexed withantigen-presenting molecules.

T Cell Antigen/epitope: An antigen or epitope that is recognized by andtriggers an immune response in a T cell (e.g., an antigen that isspecifically recognized by a T cell receptor on a T cell viapresentation of the antigen or portion thereof bound to a majorhistocompatibility complex molecule (MHC). T cells antigens generallyare proteins or peptides. This term includes peptide antigens recognizedby alpha-beta T cells or gamma-delta T cells, as well as lipid,heat-shock proteins, or other stress-induced antigens which may berecognized by gamma-delta T cells. A T cell antigen may be an antigenthat stimulates a CD8+ T cell response, a CD4+ T cell response, a gammadelta T-cell response or that stimulates a combination of these.

An “antigen presenting cell (APC)” refers to a class of cells capable ofpresenting one or more antigens in the form of peptide-MHC complexrecognizable by specific effector cells of the immune system, andthereby inducing an effective cellular immune response against theantigen or antigens being presented. Examples of professional APCs aredendritic cells and macrophages, though any cell expressing MHC Class Ior II molecules can potentially present a peptide antigen.

A “control” is a reference sample or subject used for purposes ofcomparison with a test sample or test subject. Positive controls measurean expected response and negative controls provide reference points forsamples where no or a background response is expected.

“Cord blood” has its normal meaning in the art and refers to blood thatremains in the placenta and umbilical cord after birth and containshematopoietic stem cells. Cord blood may be fresh, cryopreserved orobtained from a cord blood bank.

The term “cytokine” has its normal meaning in the art. Examples ofcytokines used in the invention include IL-2, IL-7 and IL-15.

The term “dendritic cell” or “DC” describes a diverse population ofmorphologically similar cell types found in a variety of lymphoid andnon-lymphoid tissues, see Steinman, Ann. Rev. Immunol. 9:271-296(1991).¹⁹ Some embodiments of the invention involve dendritic cells anddendritic cell precursors derived from cord blood.

The term “effector cell” describes a cell that can bind to or otherwiserecognize an antigen and mediate an immune response. Antigen-specific Tcells are effector cells.

“Human papilloma virus” is a DNA virus in the papilloma virus familythat infects humans. At least 170 subtypes are known, many of which aretransmitted by sexual contact. Types 16, 18, 31, 33, 35, 39, 45, 51, 52,56, 58, 59, 68, 73, and 82 are carcinogenic “high-risk” sexuallytransmitted HPVs and may lead to the development of cervicalintraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN),penile intraepithelial neoplasia (PIN), and/or anal intraepithelialneoplasia (AIN).^(6, 8, 20-22)

HPV is associated with common warts—HPV types 2 and 4 (most common);also types 1, 3, 26, 29, and 57 and others. Cancers and genitaldysplasia—“high-risk” HPV types are associated with cancers, notablycervical cancer, and can also cause some vulvar, vaginal, penile, analand some oropharyngeal cancers. “Low-risk” types are associated withwarts or other conditions. High-risk: 16, 18 (cause the most cervicalcancer); also 31, 33, 35, 39, 45, 52, 58, 59, and others. Plantar warts(myrmecia)—HPV type 1 (most common); also types 2, 3, 4, 27, 28, and 58and others. Anogenital warts (condylomata acuminata or venerealwarts)—HPV types 6 and 11 (most common); also types 42, 44 and others.Low-risk: 6, 11 (most common); also 13, 44, 40, 43, 42, 54, 61, 72, 81,89, and others. Flat warts—HPV types 3, 10, and 28. Butcher's warts—HPVtype 7. Heck's disease (Focal epithelial hyperplasia)—HPV types 13 and32.^(6, 8, 20-22)

An “infiltrating lymphocyte” is one that has entered a particularbiological compartment or tissue, mucosal layer (e.g., epithelium,lamina propia) or skin layer (e.g., stratum comeum, stratum lucidum,stratum granulosum, stratum spinosum, stratum basate and dermis). Theseinclude those infiltrating a neoplasm, tumor or cancer includingcervical cancer, anal, penile, oropharyngeal, vulvar, or vaginalcancers. It includes lymphocytes that have infiltrated a papilloma,including squamous cell papilloma, inverted papilloma, urothelialpapilloma, intraductal papilloma or the breast, or wart, such as aplantar or genital wart. Antigens recognized by infiltrating lymphocytesmay be selected to induce T cells having specificity for HPV relatedconditions in particular tissues.

The term “isolated” means separated from components in which a materialis ordinarily associated with, for example, an isolated cord bloodmononuclear cell can be one separated from red blood cells, plasma,and/or other components of cord blood.

“Mucosa” or mucous membranes have their conventional anatomical andphysiological meanings. These include ocular mucosal, nasal mucosa,olfactory mucosa, oral mucosa, bronchial mucosa and the lining of vocalfolds, esophageal mucosa, gastric mucosa, intestinal mucosa, analmucosa, penile mucosa, and vaginal mucosa, and endometrium which is themucosa of the uterus. Mucosa are susceptible to infection by HPV.

A “naive” T-cell or other immune effector cell is one that has not beenexposed to an antigen or to an antigen-presenting cell presenting apeptide antigen capable of activating that cell.

A “peptide library” or “overlapping peptide library” within the meaningof the application is a complex mixture of peptides which in theaggregate covers the partial or complete sequence of a protein antigen,especially those of opportunistic viruses. Successive peptides withinthe mixture overlap each other, for example, a peptide library may beconstituted of peptides 15 amino acids in length which overlap adjacentpeptides in the library by 11 amino acid residues and which can span theentire length of a protein antigen or select portions of an antigen.Peptide libraries are commercially available and may be custom-made forparticular antigens. Methods for contacting, pulsing or loadingantigen-presenting cells are well known and incorporated by reference toNgo, et al. (2014).²³

The term “precursor cell” refers to a cell which can differentiate,mature, or otherwise be transformed into another kind of cell. Forexample, a “T-cell precursor cell” can differentiate into a T-cell and a“dendritic precursor cell” can differentiate into a dendritic cell.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to humans, simians, equines,bovines, porcines, canines, felines, murines, other farm animals, sportanimals, or pets. Subjects include those in need of antigen-specific Tcells, such as those with lymphocytopenia, those who have undergoneimmune system ablation, those undergoing transplantation and/orimmunosuppressive regiments, those having naïve or developing immunesystems, such as neonates, or those undergoing cord blood or stem celltransplantation.

Generation of HPV16 E6/E7 Specific T Cells

Overlapping peptide libraries (15-mers overlapping by 11 amino acids) ofHPV16-E6 (Protein ID P03126) and HPV16-E7 (P03129) were purchased fromJPT Peptide Technologies (Berlin, Germany). Peptides were reconstitutedin dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, Mo.).

Monocytes were isolated from PBMCs by CD14 selection using MACS Beads(Miltenyi Biotec, San Diego, Calif.) and cultured in 24-well plates(1×10⁶ cells/well) in DC medium [CellGenix medium (CellGenix GmbH,Freiburg, Germany) and 1% alanyl-glutamine (GlutaMAX; Gibco LifeTechnologies, Grand Island, N.Y.)], with 800 U/mLgranulocyte/macrophage-colony stimulating factor (GM-CSF) (R&D Systems,Minneapolis, Minn.) and 1000 U/mL IL-4 (R&D Systems).

DCs were fed with GM-CSF and IL-4 on day 3. On the morning of day 5, 1ml of media was removed from the wells and 1 μL peptide mixture (E6 andE7) was added to each of the wells. At least one hour later in theafternoon of day 5, DCs were matured by adding 1 mL of DC media per wellwith the following cytokines at these final concentrations: 100 ng/mLIL-6 (R&D Systems), 10 ng/mL IL-1β (R&D Systems), 10 ng/mL TNFα (R&DSystems), 1 μg/mL prostaglandin E2 (R&D Systems), 800 U/mL GM-CSF, 1000U/mL IL-4, and 30 ng/mL LPS.

On day 7, DCs were harvested and re-suspended at 1×10⁵ cells/mL in CTLmedium [45% Click's medium (Irvine Scientific, Santa Ana, Calif.), 45%RPMI-1640 (HyClone, Logan, Utah), 10% human AB serum (GeminiBioProducts, West Sacramento, Calif.) and 1% GlutaMAX], CD14 negativePBMCs were thawed and re-suspended at 1×10⁶ cells/mL in CTL medium.Cells were stimulated with peptide-loaded DCs at a 1:10 ratio(DCs:CD14−) in CTL medium with 10 ng/mL IL-6, 10 ng/mL IL-7 (R&DSystems), 10 ng/mL IL-12 (R&D Systems) and 10 ng/mL IL-15 (R&D Systems).

Nine to twelve days after the first stimulation, T cells were harvested,counted and stimulated again at a concentration of 1×10⁶ cells/mL in CTLmedium (1×10⁶ cells/mL per well of 24-well plates) with newly preparedpeptide-loaded DCs and IL-7 and IL-15 (or alternatively with IL-7,IL-12, and IL-15, or IL-6, IL-7, IL-12, and IL-15). Cell cultures weresupplemented with IL-2 (R&D Systems) if necessary.

Seven days after the second stimulation, T cells were harvested,counted, re-suspended at 2.5×10⁵/mL and stimulated for a third time withnewly prepared peptide-loaded DCs and IL-2 and IL-15. Cell cultures weresupplemented with alternating IL-2 or IL15 (R&D Systems) if necessary.Alternatively, cells are given other antigen presenting cells mentionedabove—also loaded with peptides.

The HPV-specific T cells produced by this method were evaluated as shownby FIGS. 1-3 . These figures show that the method disclosed hereingenerates T cells from cells obtained from healthy donors not infectedby HPV that recognize HPV E6 and E7 antigens. These cells can beexpanded as described herein and exhibit cell surface markers that havea mixed phenotype (CD4 and CD8). A mixed phenotype has been shown topromote better responses from T cell infusions. Cells also express thecostimulatory receptors CD27 and CD28.

Expression of mixed CD4+CD8+ cell surface markers has been associatedwith induction of an adaptive immune response against infectiouspathogens such as those of multiple past, latent and high-levelpersistent viral infections. CD4+CD8+ T cells broaden the perception ofT cell population involved in antiviral immune responses; Nacimbene, etal., Blood 2004 104:478-486. The methods described herein generate alarge numbers of mixed CD4+CD8+ T cells that recognize HPV and maypotentially serve as superior effectors of anti-HPV immunity than Tcells having other cell surface phenotypes.

Expression of CD4+, CD45RO+ and CD27+ T cell markers is associated withdecreased persistence of HPV and with resistance to HPV infection;Rodriguez, et al., Int J Cancer. 2011 Feb. 1; 128(3): 597-607.HPV-specific T cells produced by the method disclosed herein produce maypotentially be used to protect subjects having or at risk of acquiringpersistent HPV infections. These at risk subjects include smokers,tobacco smokers or users of chewing tobacco, those with poor oralhygiene who are at increased risk of oropharyngeal cancer, those havingweakened immune systems, including diseases like HIV, those onimmunosuppressive drugs or regimens, such as cancer patients or subjectsreceiving blood transfusions or tissue transplants, and those at higherrisk of cervical cancer including women having multiple children, womenusing oral contraceptives, especially for a long term. Subjects withdiseases, disorders or conditions characterized by chronic inflammationor aged individuals (e.g., at or above 50, 60, 70, or 80 years of age)are also at higher risk of persistent HPV infection than normalsubjects.

T cells generated by the methods disclosed herein also contain a highfrequency or level of costimulatory receptors CD27 and CD28 as shown inFIG. 3 .

T cells expressing CD27 can be costimulated via CD27. Viruses are knownto modulate expression of CD27 as a means to evade the host immunesystem. Generation of T cells expressing CD27 provides a means for thesecells to induce virus-specific immunity during acute or persistent viralinfection, Welten, et al., J Virol. 2013 June; 87(12):6851-65. Themethods disclosed herein produce large numbers of T cells that recognizeHPV antigens that are CD27+ and that would be capable of costimulationvia CD27. T-cell persistence is promoted by CD27 costimulation and mayimprove clinical efficacy of adoptive immunotherapy of subjects (such asthose infected with HPV or having persistent HPV infections. Mechanismsaccounting for CD27-mediated human T-cell survival in vivo may includeupregulation of anti-apoptotic molecules, instructed CD4 help andautocrine IL-2 production by CD8⁺ T cells in vivo; Song, et al.,Oncoimmunology, 2012 Jul. 1; 1(4): 547-549.

T cells expressing CD28 can be costimulated via CD28 during the effectorphase of an anti-viral T cell response. T cells that do not express CD28or in which the CD28 receptor is blocked exhibit decreased responses tovirus and result in increased apoptosis and decreased viral clearance;Dolfi, et al., J Immunol. 2011 Apr. 15; 186(8):4599-608. The methodsdisclosed herein produce large numbers of T cells that recognize HPVantigens that are CD28+ and that would be capable of costimulation viaCD28.

The process according to the present invention is broad in scope. Forexample, it can be used to prevent conditions associated with HPV postallogeneic stem cell transplant, or it can be used to prevent relapse ofHPV malignancies like cervical carcinoma. Specific non-limitedembodiments of the invention are now further described.

In one embodiment, the invention comprises a process for producing anHPV antigen-specific T cell comprising (a) dividing mononuclear cellsfrom any cell source containing naïve immune cells into severalportions; (b) stimulating a portion of said sample with PHA or anothermitogen and with IL-2 to produce ATCs activated T cells (“ATCs”) thatserve antigen presenting functions during subsequent stimulations andoptionally treating the ATCs with radiation or another agent to inhibittheir outgrowth; (c) separating T cells and T-cell precursor cells(e.g., non-adherent cells, CD3⁺ cells, CD14⁻ cells) from dendritic cellsand dendritic precursor cells (e.g, adherent cells, CD11C⁺ or CD14⁺cells); (d) cryopreserving or otherwise reserving the T cells and T-cellprecursor cells; (e) differentiating the dendritic cells and dendriticprecursor cells in the second portion with cytokine(s) or other agent(s)that generate and mature dendritic cells and with at least one HPVpeptide antigen or HPV antigen mix to produce antigen-presentingdendritic cells that present at least one HPV peptide antigen, andoptionally treating said antigen-presenting dendritic cells withradiation or another agent sufficient to inhibit their outgrowth; (f)stimulating the cryopreserved or otherwise reserved T cells and T-cellprecursor cells from (d) with the dendritic antigen-presenting cellsproduced in (e) in the presence of any combination or all of IL-6, IL-7,IL-12 and IL-15 to produce antigen-specific T cells that recognize theat least one HPV peptide antigen; (g) stimulating antigen-specific Tcells produced by (f) with the ATCs of (b) in the presence of the atleast one HPV peptide antigen, optionally, in the presence ofpotentially artificial or alternative antigen presenting cells or otheraccessory cells in the presence of IL-2 and/or IL-15; optionally,repeating (g) one or more times; and (h) recovering antigen-specific Tcells that recognize at least one peptide antigen.

T cells the recognize HPV antigens may be further isolated or purifiedbased on cell surface markers. T cell phenotypes include cells with oneor more of the following markers: CD4+, CD8+, CD4+/CD25+, CD45RO+,CD27+, CD28+, and/or PD1. T cell phenotypes include CD4+CD8+; CD27+CD28+and CD4+, CD45RO+ and CD27+. Cells with undesired phenotypes may beremoved or separated from desired HPV recognizing T cells using methodsknown in the art.

In methods according to the invention it is unnecessary to use viablevirus to generate T cells recognizing HPV antigens. In the Examplesabove no strains of HPV or any live virus were used to produce any ofthe T cells. The antigens were from JPT: the E6 and E7 antigens wereused. Nevertheless, the methods described herein can be practiced withHPV antigens from other sources such as virus infected cell-lysate,whole virus proteins, and plasmids or DNA sequences or viral vectorsencoding the HPV antigen.

The process as described in the embodiment above may further compriseseparating mononuclear cells containing naïve T cells prior to (a). Themononuclear cells used in this process may be obtained from cord blood,from stem cells, or from other sources of cells naïve to HPV antigens ornaïve to particular HPV strains or particular HPV antigens. Thus, themononuclear cells used in this process may be obtained fromhematopoietic stem cells naïve to the at least one HPV peptide antigen;or from a sample containing stem cells, precursor T cells, or T cellsfrom a subject whose immune system is naïve to the at least one HPVpeptide antigen.

A process according to the invention may comprise (b) stimulating afirst portion of said sample with PHA and with and IL-2 to produceactivated T cells (“ATCs”). These ATCs may be cryopreserved or otherwisebanked for later use or may be used immediately. Preferably, the ATCsare used fresh and mixed in with antigen-specific T cells produced in(f) without the need to cryopreserve either the ATCs or theantigen-specific T cells. For example, PHA blasts prepared in (b) can beused 14-16 days after initiation of the process to provide a secondstimulation to the antigen-specific T cells produced in (f).

While those skilled in the art may select a suitable number of cells foruse in a process according to the invention, such a process may comprisestimulating about 1 to 20 million, preferably 5-15 million, mostpreferably about 8-12 million, mononuclear cord blood cells with PHA andIL-2 in (b). Those of skill in the art may adjust the numbers of cellsor other process conditions as necessary to scale up or scale down theprocess producing T cells recognizing HPV peptide antigens. In a processaccording to the invention, (b) may comprise producing T-blasts,B-blasts, lymphoblastoid cells, or CD3-CD28 blasts instead of PHA blastsas activated T cells.

Such a process may use T cells and/or T cell precursor cells that areseparated from dendritic cells and dendritic precursor cells byculturing the second portion via plastic adherence under conditionssufficient for cells in the second portion to adhere to the cell cultureplate or device and then removing T cells and T cell precursor cellsfrom the cell culture plate or device and recovering the dendritic cellsand dendritic precursor cells attached to the solid medium.Alternatively, these two populations of cells may be separatedmagnetically, by the use of antibodies that specifically recognize eachpopulation, by CD14-based selection (e.g., using MAC beads) or by otherknown methods of cell sorting. The separate populations of cells may becryopreserved or otherwise banked for later use, or may be usedimmediately to produce T cells or dendritic cells. These populations mayalso be cryopreserved or otherwise banked after subsequent treatmentsteps described herein that produce mature dendritic cells loaded withpeptide antigens or antigen-specific T cells.

In a process of the invention in (e) the dendritic cells and dendriticprecursor cells can be grown in the presence of cytokines necessary fordifferentiation and maintenance of dendritic cells, including but notlimited to IL-4 and GM-CSF; and in (e) the dendritic cells and dendriticprecursor cells may be allowed to undergo maturation with a dendriticcell-maturing cytokine or agent selected from the group consisting of,but not limited to, one or more of the following LPS, TNF-alpha, IL-1beta, IL-6, PGE-1, PGE-2, and other immune adjuvants (e.g.oligonucleotides), along with IL-4 and GM-CSF.

In some embodiments of the invention in or prior to (f) the T cells or Tcell precursor cells may be treated to expand CD45RA positive cells; orin or prior to, (f) the T cells and T cell precursors may be treated todeplete CD45RO positive cells. Other T cells with particular markers maybe treated to expand or deplete HPV-recognizing T cells havingparticular cell surface phenotypes, such as any of those phenotypesdescribed herein.

In a process according to the invention, the at least one antigen maycomprise a series of overlapping peptides spanning an entire HPV proteinor HPV antigen. In some embodiments the antigen-specific T cells may beselected to recognize at least one antigen of an opportunistic HPV thatis acquired nosocomially or iatrogenically or that is transmitted to asubject in a hospital (e.g., a hospital acquired infection) or by aparticular mode of invention, such as by direct contact with a mucousmembrane, skin, or through sexual contact. The at least one peptideantigen may comprise a HPV antigen or peptide library, such as an HPVE1-E5 antigen or peptide library; an HPV E6 or E7 antigen or peptidelibrary; an HPV L1 or L2 antigen or peptide library; or mixturesthereof.

The at least one peptide antigen may be one recognized by a T-cellisolated from a site, tissue or cell infected with HPV.

Another embodiment of the invention is directed to a compositioncomprising antigen-specific T cells produced by a process according tothe invention. Such antigen specific T cells may recognize one, two,three, four, five or more HPV strains, serotypes, antigens or epitopes.Such a composition may comprise mononuclear cells isolated fromdifferent cell sources, which preferably mostly comprise but are notlimited to samples containing naïve immune cells, antigen presentingcells derived from these same sources, such as dendritic cells, PHAblasts—which contain in them PHA or another mitogen, IL-2, and a mediumthat maintains the viability of said cells, and, optionally, K562 cellsor other non-autologous cells, including artificial or alternativeantigen presenting cells, that costimulate T cells, wherein, optionally,said cells have been treated to prevent outgrowth.

In another embodiment the comprising may comprise T cells and T cellprecursor cells (e.g., non-adherent cells, CD3⁺ cells, CD14⁻ cells) thathave been separated from dendritic cells and dendritic precursor cells(e.g, adherent cells, CD11C⁺ or CD14⁺ cells) and that are naïve to anHPV peptide antigen; cytokines including but not limited to at least oneof IL-2, IL-6, IL-7, IL-12, and IL-15, at various points in the cultureperiod; and a medium that maintains the viability of said T cells andT-cell precursor cells.

Other embodiments of the invention include a bank or cell storagefacility which contains one or more samples of T cells that recognizeHPV peptide antigens produced by the process of embodiment 1 incombination with a storage or freezing medium; wherein said one or moresamples is optionally associated, identified or indexed by informationdescribing its source, including full or partial DNA sequenceinformation, information describing its histocompatibility, includingmajor and/or minor histocompatibility antigens or markers, and/orinformation about the peptide antigens it contains or recognizes. Suchan antigen-specific T cell bank may comprise multiple samples of cryo-or otherwise preserved viable HPV antigen-specific T cells produced bythe process of embodiment 1.

Another embodiment of the invention constitutes a kit comprising T cellsand T cell precursor cells (e.g., non-adherent cells, CD3⁺ cells, CD14⁻cells) that have been separated from dendritic cells and dendriticprecursor cells (e.g, adherent cells, CD11C⁺ or CD14⁺ cells) and thatare naïve to an HPV peptide antigen; cytokines including but not limitedto at least one of IL-2, IL-6, IL-7, IL-12, and IL-15; a medium thatmaintains the viability of said T cells and T-cell precursor cells;dendritic or dendritric precursor cells that have been separated fromsaid T cells and T cell precursor cells; a medium that maintains theviability of said dendritic or dendritic precursor cells; K562 cells,artificial or alternative antigen presenting cells, other non-autologouscells that costimulate T cells, optionally treated to prevent outgrowth,and at least one HPV peptide antigen or mixture of HPV peptides.

The foregoing description discloses particular embodiments. As will beunderstood by those skilled in the art, the approaches, methods,techniques, materials, devices, and so forth disclosed herein may beembodied in additional embodiments as understood by those of skill inthe art, it is the intention of this application to encompass andinclude such variation. Accordingly, this description is illustrativeand should not be taken as limiting the scope of the following claims.Citations herein are incorporated by reference especially for thepurposes of specifically describing the referenced subject matter or thesubject matter in the same or adjoining paragraphs or sections.

REFERENCES

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We claim:
 1. A method for treating or preventing an infection by humanpapillomavirus (HPV) in a subject, comprising: administering to thesubject an HPV antigen-specific T cell population, wherein the HPVantigen-specific T cell population is prepared by a method comprising:(a) dividing mononuclear cells from a donor source containing immunecells naïve to an HPV antigen into a CDI4-positive cell population and aCD14-negative cell population; (b) culturing a first portion of theCD14-positive cell population in a medium comprisinggranulocyte/macrophage-colony stimulating factor (GM-CSF) and IL-4 toproduce a first population of dendritic cells; (c) culturing the firstpopulation of dendritic cells produced in step (b) in a mediumcomprising peptides derived from two or more HPV antigens; (d) culturingthe first population of dendritic cells from step (c) in a mediumcomprising IL-6, IL-1β, TNFα, prostaglandin E2, GM-CSF, IL-4, and LPS;(e) culturing the CD14-negative cell population with the firstpopulation of dendritic cells from step (d) in a medium comprising IL-6,IL-7, IL-12, and IL-15 to produce an HPV-antigen specific T-cellpopulation; (f) culturing the HPV-antigen specific T-cell populationfrom step (e) with a second population of dendritic cells in a mediumcomprising IL-7 and IL-15, wherein the second population of dendriticcells are generated from a second portion of the CD-14 positive cellpopulation as provided in steps (b)-(d); (g) culturing the HPV-antigenspecific T-cell population from step (f) with a third population ofdendritic cells in a medium comprising IL-2 and IL-15, wherein the thirdpopulation of dendritic cells are generated from a third portion of theCD-14 positive cell population as provided in steps (b)-(d); and, (h)recovering the HPV-antigen specific T cell population from step (g);wherein the recovered HPV-antigen specific T-cell population comprises amixed phenotype of CD4+ and CD8+ T-cells, and, wherein the recoveredHPV-antigen specific T-cell population comprises CD27+ and CD28+T-cells.
 2. The method of claim 1, wherein the two or more HPV antigensare selected from the group consisting of E1, E2, E3, E4, E5, E6, E7,L1, and L2.
 3. The method of claim 2, wherein the two or more HPVantigens comprise E6 and E7.
 4. The method of claim 1, wherein thepeptides derived from the two or more HPV antigens are from overlappingpeptide libraries.
 5. The method of claim 1, wherein the medium of step(f) further comprises IL-6 or IL-12.
 6. The method of claim 1, whereinthe medium of step (f) further comprises IL-6 and IL-12.
 7. The methodof claim 1, wherein the medium of step (f) further comprises IL-2. 8.The method of claim 5, wherein the medium of step (f) further comprisesIL-2.
 9. The method of claim 6, wherein the medium of step (f) furthercomprises IL-2.
 10. The method of claim 1, wherein the medium of step(g) further comprises IL-2 or IL-15.
 11. The method of claim 1, whereinthe medium of step (g) is supplemented with IL-2 and IL-15, wherein theIL-2 and IL-15 are added to the medium in an alternating schedule.
 12. Acomposition comprising an HPV antigen-specific T cell population and amedium that maintains the viability of the T cell population, whereinthe HPV antigen-specific T cell population is prepared by a methodcomprising: (a) dividing mononuclear cells from a donor sourcecontaining immune cells naïve to an HPV antigen into a CD14-positivecell population and a CD14-negative cell population; (b) culturing afirst portion of the CD14-positive cell population in a mediumcomprising granulocyte/macrophage-colony stimulating factor (GM-CSF) andIL-4 to produce a first population of dendritic cells; (c) culturing thefirst population of dendritic cells produced in step (b) in a mediumcomprising peptides derived from two or more HPV antigens; (d) culturingthe first population of dendritic cells from step (c) in a mediumcomprising IL-6, IL-1β, TNFα, prostaglandin E2, GM-CSF, IL-4, and LPS;(e) culturing the CD14-negative cell population with the firstpopulation of dendritic cells from step (d) in a medium comprising IL-6,IL-7, IL-12, and IL-15 to produce an HPV-antigen specific T-cellpopulation; (f) culturing the HPV-antigen specific T-cell populationfrom step (e) with a second population of dendritic cells in a mediumcomprising IL-7 and IL-15, wherein the second population of dendriticcells are generated from a second portion of the CD-14 positive cellpopulation as provided in steps (b)-(d); (g) culturing the HPV-antigenspecific T-cell population from step (f) with a third population ofdendritic cells in a medium comprising IL-2 and IL-15, wherein the thirdpopulation of dendritic cells are generated from a third portion of theCD-14 positive cell population as provided in steps (b)-(d); and, (h)recovering the HPV-antigen specific T cell population from step (g);wherein the recovered HPV-antigen specific T-cell population comprises amixed phenotype of CD4+ and CD8+ T-cells, and, wherein the recoveredHPV-antigen specific T-cell population comprises CD27+ and CD28+T-cells.
 13. A method for treating or preventing an infection by humanpapillomavirus (HPV) in a subject, comprising: administering to thesubject an HPV antigen-specific T cell population, wherein the HPVantigen-specific T cell population is prepared by a method comprising:(a) dividing mononuclear cells from a donor source containing immunecells naïve to an HPV antigen into a CD14-positive cell population and aCD14-negative cell population; (b) culturing a first portion of theCD14-positive cell population in a medium comprisinggranulocyte/macrophage-colony stimulating factor (GM-CSF) and IL-4 toproduce a first population of dendritic cells; (c) culturing the firstpopulation of dendritic cells produced in step (b) in a mediumcomprising the peptides derived from two or more HPV antigens; (d)culturing the first population of dendritic cells from step (c) in amedium comprising IL-6, IL-1β, TNFα, prostaglandin E2, GM-CSF, IL-4, andLPS; (e) culturing a first portion of the CD14-negative cell populationwith the first population of dendritic cells from step (d) in a mediumcomprising IL-6, IL-7, IL-12, and IL-15 to produce an HPV-antigenspecific T-cell population; (f) culturing a second portion of theCD14-negative cell population in a medium with phytohemagglutinin andIL-2 to produce a first population of activated T-cells (ATCs); (g)culturing the HPV-antigen specific T-cell population from step (e) withthe first population of ATCs from step (f) in a medium comprising thetwo or more HPV antigens, IL-2 or IL-15, or IL-2 and IL-15; (h)culturing the HPV-antigen specific T-cell population from step (g) witha second population of ATCs in a medium comprising the two or more HPVantigens, IL-2 or IL-15, or IL-2 and IL-15; wherein the secondpopulation of ATCs is generated from a third portion of the CD-14negative cell population as provided in step (f); and, (i) recoveringthe HPV-antigen specific T cell population from step (h); wherein therecovered HPV-antigen specific T-cell population comprises a mixedphenotype of CD4+ and CD8+ T-cells, and, wherein the recoveredHPV-antigen specific T-cell population comprises CD27+ and CD28+T-cells.
 14. The method of claim 13, wherein the two or more HPVantigens are selected from the group consisting of E1, E2, E3, E4, E5,E6, E7, L1, and L2.
 15. The method of claim 13, wherein the two or moreHPV antigens comprise E6 and E7.
 16. The method of claim 13, wherein thepeptides derived from the two or more HPV antigens are from overlappingpeptide libraries.
 17. The method of claim 13, wherein the medium ofstep (g) is supplemented with IL-2.
 18. The method of claim 13, whereinthe medium of step (g) is supplemented with IL-15.
 19. The method ofclaim 13, wherein the medium of step (g) is supplemented with IL-2 andIL-15.
 20. The method of claim 13, wherein the culture of step (g)further comprises K562 cells.
 21. The method of claim 13, wherein theculture of step (h) further comprises K562 cells.
 22. A compositioncomprising an HPV antigen-specific T cell population and a medium thatmaintains the viability of the T cell population, wherein the HPVantigen-specific T cell population is prepared by a method comprising:(a) dividing mononuclear cells from a donor source containing immunecells naïve to an HPV antigen into a CD14-positive cell population and aCD14-negative cell population; (b) culturing a first portion of theCD14-positive cell population in a medium comprisinggranulocyte/macrophage-colony stimulating factor (GM-CSF) and IL-4 toproduce a first population of dendritic cells; (c) culturing the firstpopulation of dendritic cells produced in step (b) in a mediumcomprising the peptides derived from two or more HPV antigens; (d)culturing the first population of dendritic cells from step (c) in amedium comprising IL-6, IL-1β, TNFα, prostaglandin E2, GM-CSF, IL-4, andLPS; (e) culturing a first portion of the CD14-negative cell populationwith the first population of dendritic cells from step (d) in a mediumcomprising IL-6, IL-7, IL-12, and IL-15 to produce an HPV-antigenspecific T-cell population; (f) culturing a second portion of theCD14-negative cell population in a medium with phytohemagglutinin andIL-2 to produce a first population of activated T-cells (ATCs); (g)culturing the HPV-antigen specific T-cell population from step (e) withthe first population of ATCs from step (f) in a medium comprising thetwo or more HPV antigens, IL-2 or IL-15, or IL-2 and IL-15; (h)culturing the HPV-antigen specific T-cell population from step (g) witha second population of ATCs in a medium comprising the two or more HPVantigens, IL-2 or IL-15, or IL-2 and IL-15; wherein the secondpopulation of ATCs is generated from a third portion of the CD-14negative cell population as provided in step (f); and, (i) recoveringthe HPV-antigen specific T cell population from step (h); wherein therecovered HPV-antigen specific T-cell population comprises a mixedphenotype of CD4+ and CD8+ T-cells, and, wherein the recoveredHPV-antigen specific T-cell population comprises CD27+ and CD28+T-cells.
 23. A method for treating or preventing an infection by humanpapillomavirus (HPV) in a subject, comprising: administering to thesubject an HPV antigen-specific T cell population, wherein the HPVantigen-specific T cell population is prepared by a method comprising:(a) dividing mononuclear cells from a naïve donor source containingimmune cells naïve to an HPV antigen into a CD14-positive cellpopulation and a CD14-negative cell population; (b) culturing a firstportion of the CD14-positive cell population in a medium comprisinggranulocyte/macrophage-colony stimulating factor (GM-CSF) and IL-4 toproduce a first population of dendritic cells; (c) culturing the firstpopulation of dendritic cells produced in step (b) in a mediumcomprising peptides derived from two or more HPV antigens; (d) culturingthe first population of dendritic cells from step (c) in a mediumcomprising IL-6, IL-1β, TNFα, prostaglandin E2, GM-CSF, IL-4, and LPS;(e) culturing the CD I4-negative cell population with the firstpopulation of dendritic cells from step (d) in a medium comprising IL-6,IL-7, IL-12, and IL-15 to produce an HPV-antigen specific T-cellpopulation; (f) culturing the HPV-antigen specific T-cell populationfrom step (e) with a second population of dendritic cells in a mediumcomprising IL-7 and IL-15, wherein the second population of dendriticcells are generated from a second portion of the CD-14 positive cellpopulation as provided in steps (b)-(d); (g) culturing the HPV-antigenspecific T-cell population from step (I) with a third population ofdendritic cells in a medium comprising IL-2 and IL-15, wherein the thirdpopulation of dendritic cells are generated from a third portion of theCD-14 positive cell population as provided in steps (b)-(d); and, (h)recovering the HPV-antigen specific T cell population from step (g);wherein the recovered HPV-antigen specific T-cell population comprises amixed phenotype of CD4+ and CD8+ T-cells, and, wherein the recoveredHPV-antigen specific T-cell population comprises CD27+ and CD28+T-cells.
 24. The method of claim 23, wherein in (e) cytokines in themedium consist of IL-6, IL-7, IL-12, and IL-15.
 25. The method of claim23, wherein in (f) cytokines in the medium consist of IL-7 and IL-15.26. A composition comprising an HPV antigen-specific T cell population,wherein the HPV antigen-specific T cell population is prepared by amethod comprising: (a) dividing mononuclear cells from a naïve donorsource containing immune cells naïve to an HPV antigen into aCD14-positive cell population and a CD14-negative cell population; (b)culturing a first portion of the CD14-positive cell population in amedium comprising granulocyte/macrophage-colony stimulating factor(GM-CSF) and IL-4 to produce a first population of dendritic cells; (c)culturing the first population of dendritic cells produced in step (b)in a medium comprising peptides derived from two or more HPV antigens;(d) culturing the first population of dendritic cells from step (c) in amedium comprising IL-6, IL-1β, TNFα, prostaglandin E2, GM-CSF, IL-4, andLPS; (e) culturing the CD14-negative cell population with the firstpopulation of dendritic cells from step (d) in a medium comprising IL-6,IL-7, IL-12, and IL-15 to produce an HPV-antigen specific T-cellpopulation; (f) culturing the HPV-antigen specific T-cell populationfrom step (e) with a second population of dendritic cells in a mediumcomprising IL-7 and IL-15, wherein the second population of dendriticcells are generated from a second portion of the CD-14 positive cellpopulation as provided in steps (b)-(d); (g) culturing the HPV-antigenspecific T-cell population from step (f) with a third population ofdendritic cells in a medium comprising IL-2 and IL-15, wherein the thirdpopulation of dendritic cells are generated from a third portion of theCD-14 positive cell population as provided in steps (b)-(d); and, (h)recovering the HPV-antigen specific T cell population from step (g);wherein the recovered HPV-antigen specific T-cell population comprises amixed phenotype of CD4+ and CD8+ T-cells, and, wherein the recoveredHPV-antigen specific T-cell population comprises CD27+ and CD28+T-cells.